Members of the small heat-shock protein (sHSP) superfamily are ubiquitous molecular chaperones that participate in physiological processes underlying stress tolerance, longevity and aging, and apoptosis. Of the ten sHSP identified in humans, alphaA- and alphaB-crystallin are resident lens proteins that maintain its optical properties whereas alphaB-crystallin and Hsp27 are abundant in muscle tissues where they protect against stressful conditions such as ischemia. Inherited mutations in alphaA- and alphaB-crystallin are linked to autosomal dominant cataract in the lens and familial desminopathy in the heart. The long-term goal of this grant is to elucidate the molecular basis of the chaperone activity of the alpha-crystallins so that we may better define their roles in health and disease. In the last funding period, biochemical and structural studies have significantly advanced our understanding of the mechanisms underlying recognition and binding. sHSP have sequence and structural elements, "stability sensors", that allow them to bind various aggregation-prone excited states of target proteins. The exposure of these sensors is controlled by a switch encoded at the level of the oligomeric structure and activated in response to stress stimuli. These developments are harnessed to propose a novel hypothesis for the molecular basis of congenital cataract linked to mutations in alphaA- and alphaB-crystallin. Through their effects on the mechanisms that regulate activation, the mutations cause a toxic enhancement of binding which transforms these proteins into unfoldases. To test this hypothesis we will carry out: 1) Structure-function studies to identify the sequence features that control binding and activation and thus confer predisposition to the deleterious effects of these mutations, 2) hermodynamic and kinetics studies to characterize the binding of cataract-linked mutants to a model substrate, and 3) Structural analysis to link the functional consequences to changes in the oligomeric equilibrium characteristic of these proteins. Central to our experimental paradigm is a novel chaperone assay that allows the dissection of recognition and binding events as well as the measurement of binding constants and stoichiometries. High throughput technologies will facilitate random and site-directed modifications and the construction of chimeras in the context of a genomic perspective on sHSP structure-function. Reporter group spectroscopic approaches will be used to provide insight into the structural basis of recognition and binding.